Astrocyte Dysfunction in Rett Syndrome Abstract Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. The disease is complex, as all key cell types (neurons, astrocytes, microglia, and oligodendrocytes) in the brain have been shown to contribute to the disease etiology. To fully understand the disease mechanism and develop effective treatments, it is essential to define the key phenotypes, link the phenotypes with loss of MeCP2 function, and reveal the consequences of the phenotypes in each cell type; and to study how these cells interact. While earlier research mostly focused on neuronal dysfunction in RTT, more recent studies have clearly demonstrated that astrocytes express MeCP2, loss of MeCP2 in astrocytes causes neuronal defects, and restoring MeCP2 expression to normal in astrocytes alleviates disease symptoms. Although a few studies have reported gene expression changes and phenotypes in Mecp2 mutant mouse astrocytes, it is not clear how these alterations contribute to RTT pathogenesis either by directly changing astrocyte functions or by indirectly changing neuronal functions. Therefore, it is necessary to systematically investigate the astrocyte cell autonomous phenotypes, the underlying mechanism of the phenotypes, and the functional consequence of the phenotypes. We have discovered significant changes in cytosolic calcium homeostasis in RTT astrocytes in the absence and presence of neurons, revealed potential molecular and cellular mechanisms underlying the abnormal calcium homeostasis, and identified major functional consequence of this astrocyte cell autonomous phenotype on neighboring neurons and the neural network. Here, we propose to 1) dissect the cell type specific contribution to the phenotypes of increase TRPC4 expression and abnormal calcium activity in astrocytes, the phenotype of excessive activation of extrasynaptic NMDA receptor (eNMDAR) in neighboring neurons, and the phenotype of increased network excitability in RTT mouse models; 2) reveal the functional significance of abnormal calcium activity in RTT disease progression; and 3) investigate the contribution of TRPC4 to the phenotype of abnormal calcium homeostasis in RTT astrocytes, and reveal the functional significance of ectopic TRPC4 expression in RTT disease progression. Our study employs various RTT mouse models (germline knockout mice, cell type specific conditional knockout mice, and cell type specific conditional reactivation mice), the RTT patient- specific induced pluripotent stem cell (iPSC) model, and genetically engineered human embryonic stem cell (hESC) model. By combining the strengths from these complementary models, we can cross-validate our findings between species and between in vitro and in vivo, therefore generating more valid insights. The successful completion of this project will significantly advance the understanding of RTT disease mechanism and facilitate future development of therapies to treat RTT.